Kimberly Burns

Endoplasmic Reticulum Form of Calreticulin Modulates Glucocorticoid-sensitive Gene Expression

Calreticulin is a ubiquitously expressed Ca2+-binding protein of the endoplasmic reticulum (ER), which inhibits DNA binding in vitro and transcriptional activation in vivo by steroid hormone receptors. Transient transfection assays were carried out to investigate the effects of different intracellular targeting of calreticulin on transactivation mediated by glucocorticoid receptor. BSC40 cells were transfected with either calreticulin expression vector (ER form of calreticulin) or calreticulin expression vector encoding calreticulin minus leader peptide, resulting in cytoplasmic localization of the recombinant protein. Transfection of BSC40 cells with calreticulin expression vector encoding the ER form of the protein led to 40-50% inhibition of the dexamethasone-sensitive stimulation of luciferase expression. However, in a similar experiment, but using the calreticulin expression vector encoding cytoplasmic calreticulin, dexamethasone-stimulated activation of the luciferase reporter gene was inhibited by only 10%. We conclude that the ER, but not cytosolic, form of calreticulin is responsible for inhibition of glucocorticoid receptor-mediated gene expression. These effects are specific to calreticulin, since overexpression of the ER lumenal proteins (BiP, ERp72, or calsequestrin) has no effect on glucocorticoid-sensitive gene expression. The N domain of calreticulin binds to the DNA binding domain of the glucocorticoid receptor in vitro; however, we show that the N+P domain of calreticulin, when synthesized without the ER signal sequence, does not inhibit glucocorticoid receptor function in vivo. Furthermore, expression of the N domain of calreticulin and the DNA binding domain of glucocorticoid receptor as fusion proteins with GAL4 in the yeast two-hybrid system revealed that calreticulin does not interact with glucocorticoid receptor under these conditions. We conclude that calreticulin and glucocorticoid receptor may not interact in vivo and that the calreticulin-dependent modulation of the glucocorticoid receptor function may therefore be due to a calreticulin-dependent signaling from the ER.

The high-affinity calcium binding protein of sarcoplasmic reticulum. Tissue distribution, and homology with calregulin

The 55-kDa high-affinity calcium binding protein (HACBP) was first identified and isolated from skeletal muscle sarcoplasmic reticulum (SR). Using polyclonal antibodies raised against the HACBP isolated from skeletal muscle we have identified this protein in cardiac and smooth muscle as well as in non-muscle cells. Although the 55-kDa protein has a size, properties and localization similar to that of calsequestrin, the two proteins are immunologically distinct. The NH2-terminal sequence of uterine HACBP is also completely different from that of calsequestrin but it is identical to that of rabbit liver calregulin, a recently identified calcium binding protein. Indirect immunofluorescence staining of frozen sections and culture cells from a variety of tissues shows that the 55-kDa protein localizes predominantly to junctional SR and T-tubule areas in skeletal muscle, to SR in smooth and cardiac muscle cells, and to ER in a variety of non-muscle cells. These data show that the protein is present in a wide variety of tissues and suggest that it is a protein common for both sarcoplasmic and endoplasmic reticulum membranes.

Calreticulin: from Ca2+ binding to control of gene expression

Calreticulin is a highly conserved Ca(2+)-binding/storage protein of the endoplasmic reticulum (ER). Recently, it has been shown to play a role in the control of gene expression by interacting with the DNA-binding domain of various steroid receptors. How does this ER protein gain access to the nuclear steroid receptors? We propose that calreticulin undergoes unique intracellular trafficking that allows it to colocalize with and bind to steroid receptors.

Expression and purification of recombinant and native calreticulin

Calreticulin is a 60-kDa Ca(2+)-binding protein of the endo(sarco)plasmic reticulum membranes of a variety of cellular systems. The protein binds approximately 25 mol of Ca2+ with low affinity and approximately 1 mol of Ca2+ with high affinity and is believed to be a site for Ca2+ binding/storage in the lumen of the endo(sarco)plasmic reticulum. In the present study, we describe purification procedures for the isolation of recombinant and native calreticulin. Recombinant calreticulin was expressed in Escherichia coli, using the glutathione S-transferase fusion protein system, and was purified to homogeneity on glutathione-Sepharose followed by Mono Q FPLC chromatography. A selective ammonium sulfate precipitation method was developed for the purification of native calreticulin. The protein was purified from ammonium sulfate precipitates by diethylaminoethyl-Sephadex and hydroxylapatite chromatography procedures, which eliminates the need to prepare membrane fractions. The purification procedures reported here for recombinant and native calreticulin yield homogeneous preparations of the proteins, as judged by the HPLC reverse-phase chromatography and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified native and recombinant calreticulin were identified by their NH2-terminal amino acid sequences, by their Ca2+ binding properties, and by their reactivity with anticalreticulin antibodies.

Interactions of calreticulin with proteins of the endoplasmic and sarcoplasmic reticulum membranes

The ability of [125I]calreticulin to bind to membrane fractions isolated from different muscle and non‐muscle tissues was examined by a protein overlay technique. Specific [125AI]calreticulin binding proteins were detected in rat liver smooth and rough endoplasmic reticulum and Golgi, in canine pancreatic microsomes, and in rabbit skeletal muscle sarcoplasmic reticulum. These proteins were confined only to membranes that contain calreticulin; they were not found in rat liver mitochondria or cytosol. [125I]Calreticulin binds to a 50‐kDa protein and a number of lower Mr (20,000–38,000) endoplasmic reticulum membrane proteins and to 30‐kDa protein in skeletal muscle sarcoplasmic reticulum. Full‐length calreticulin and the carboxyl‐terminal region (C‐domain) of the protein both competed with [125I]calreticulin for binding to the membrane proteins. Binding of [125I]calreticulin to pancreatic microsomes was also partially inhibited by the N‐domain and to a lesser extent by the P‐domain of the protein. We conclude that calreticulin interacts with the endoplasmic reticulum membrane proteins mainly through its carboxyl‐terminal domain and that the endoplasmic and sarcoplasmic reticulum membranes may contain different calreticulin binding proteins.

Calreticulin modulates the in vitro DNA binding but not the in vivo transcriptional activation by peroxisome proliferator-activated receptor/retinoid X receptor heterodimers.

Calreticulin is a ubiquitous calcium binding/storage protein found primarily in the endoplasmic reticulum. Calreticulin has been shown to inhibit DNA binding and transcriptional activation by glucocorticoid and androgen hormone receptors by binding to the conserved sequence KXFF(K/R)R, present in the DNA-binding domains of all known members of the steroid/nuclear hormone receptor superfamily. To determine whether calreticulin might be a general regulator of hormone-responsive pathways, we examined its effect on DNA binding in vitro and transcriptional activation in vivo by heterodimers of the peroxisome proliferator-activated receptor (PPAR) and the 9-cis retinoic acid receptor (RXR alpha). We show here that purified calreticulin inhibits the binding of PPAR/RXR alpha heterodimers and of other nuclear hormone receptors, to peroxisome proliferator-responsive DNA elements in vitro. However, overexpression of calreticulin in transiently transfected cultured cells had little or no effect on transactivation mediated by PPAR/RXR alpha. Therefore, while calreticulin inhibits the binding of both nuclear and steroid hormone receptors to cognate response elements in vitro, our findings suggest that calreticulin does not necessarily play an important role in the regulation of all classes of hormone receptors in vivo.

Calreticulin: A Granule-Protein by Default or Design?

Calreticulin is a major calcium binding protein normally found in the lumen of the endoplasmic reticulum (ER). When T lymphocytes were activated we observed an increase in the levels of calreticulin mRNA and protein (Burns et al. 1992). Initially we thought this induction might relate to the changes in intracellular Ca2+ levels that have been associated with signal transduction, ultimately leading to specific gene transcription and cell activation. However, subsequent experiments revealed that calreticulin itself can also directly influence patterns of steroid hormone-dependent gene expression by binding to the receptor protein (Burns et al. 1994a). This left us with somewhat of a conundrum: in order to influence gene expression calreticulin would have to find its way from the ER to the nucleus, possibly via the cytoplasm. We were very surprised to discover that, when we immunolocalized calreticulin in activated cytotoxic T cells (CTLs) the major positive organelles were none other than our old friends the cytoplasmic granules. Concomitantly a report appeared on the NH2-terminal sequence analysis of a 60 kDa granule-associated protein that copurifies with perforin. The peptide was identical to calreticulin (Dupuis et al. 1993).

Calreticulin inhibits glucocorticoid- but not cAMP-sensitive expression of tyrosine aminotransferase gene in cultured McA-RH7777 hepatocytes.

Calreticulin is a ubiquitously expressed Ca2+ binding protein of the endoplasmic reticulum which inhibits DNA binding and transcriptional activation by steroid hormone receptors. In this study the effects of calreticulin on tyrosine aminotransferase (TAT) gene expression in cultured McA–RH7777 hepatocytes was investigated. McA–RH7777 cells were stably transfected with calreticulin expression vector to generate cells overexpressing the protein. The transcriptional activity of the TAT gene, which is glucocorticoid–sensitive and cAMP–dependent, was investigated in the mock transfected McA–RH7777 and in cells overexpressing calreticulin (designated McA–11 and McA–17). In the presence of dexamethasone or the cAMP analog (CTP–cAMP) expression of the TAT gene was induced in mock transfected McA–RH7777 cells by approximately 4.5 and 5 fold, respectively. In McA–11 and McA–17 cells, overexpressing calreticulin, glucocorticoi ever, the CTP–cAMP–dependent expression of the TAT gene was not affected. The ability of calreticulin to inhibit glucocorticoid–sensitive TAT gene expression but not the cAMP–dependent expression of the gene suggests that the protein affects specifically the action of transcription pathways involving steroid receptors or transcription factors containing KxFF(K/R)R–like motifs. Calreticulin may play an important role in the regulation of glucocorticoid–sensitive pathway of expression of the hepatocytes specific genes during development.